全氟化碳对脂多糖诱导的肺泡液体清除能力下调的保护作用

发布时间：2018-06-20 19:27

本文选题：急性肺损伤/急性呼吸窘迫综合征 + II型肺泡上皮细胞　；参考：《中国人民解放军医学院》2013年硕士论文

【摘要】：目的 ALI/ARDS患者肺泡液体清除相关离子通道表达和功能活性下降，肺泡液体清除能力受损，肺泡腔内液体积聚增加，是导致呼吸窘迫的直接原因，与疾病预后差相关。通过改善肺泡液体清除能力，可能是未来救治ALI/ARDS的新思路。本研究探讨全氟化碳对脂多糖诱导的肺泡液体清除相关离子通道表达及功能下调的保护作用。方法（1）利用超声细胞破碎仪制备PFC-in-DMEN；采用不同体积比的PFC-in-DMEM（0%，10%，30%，50%）作用A549细胞0.5h、1h、2h、4h，优化PFC作用最佳浓度和作用时间;（2）用LPS（10ug/ml）干预A549细胞，建立肺损伤细胞模型；（3）A549细胞分别采用DMEM培养基、LPS（10ug/ml）、10%PFC-in-DMEM、10%PFC-in-DMEM~+LPS（10ug/ml）干预4h，采用实时定量PCR（qPCR）检测ENaC-α、β、γ,Na~+-K~+-ATPase-α1,β1,CNG1-α和AQP3mRNA的变化;western-blot检测ENaC-α、Na~+-K~+-ATPase-α1的蛋白表达变化；采用细胞Na~+-K~+-ATPase酶活性检测试剂盒分析A549细胞Na~+-K~+-ATPase活性。结果（1）10%PFC作用A549细胞4h ENaC-α和Na~+-K~+-ATPase-α1mRNA表达的水平最高；（2）LPS(10ug/ml)增加1L-1β、IL-6和TNF-α的mRNA表达，4h达到最高峰，，6h呈下降趋势，但仍高于对照组。LPS作用0.5h时ENaC-α和Na~+-K~+-ATPase-α1mRNA轻度上升，1h后呈逐渐下降趋势，但均无统计学差异，4h下降最明显（分别P=0.037，P=0.046）；（3）与对照组比较，LPS组ENaC-α、β,Na~+-K~+-ATPase-α1、β1的mRNA的表达和6h的ENaC-α、Na~+-K~+-ATPase-α1蛋白的表达明显降低，而ENaC-γ、CNG1-α、和AQP3无明显降低。（4）与LPS比较，PFC~+LPS作用4h能显著上调ENaC-α、γ,Na~+-K~+-ATPase-α1,β1和CNG1-α的mRNA表达，显著上调ENaC-α、Na~+-K~+-ATPase-α1的蛋白表达；（5）与LPS比较，PFC~+LPS作用4h能显著上调Na~+-K~+-ATPase活性（P=0.045）。结论 1．10ug/ml LPS能导致A549细胞产生大量IL-1β、IL-6和TNF-α等前炎症因子，并使肺泡液体清除相关离子通道ENaC-α、β,Na~+-K~+-ATPase-α1、β1的mRNA的表达和6h的ENaC-α、Na~+-K~+-ATPase-α1蛋白的表达下降，表明ALI细胞模型成功，并可用于ALI时肺泡液体清除相关细胞研究。 2. PFC能直接提高正常A549细胞肺泡液体清除相关离子通道ENaC-γ和CNG1αmRNA和ENaC-α和Na~+-K~+-ATPase-α1蛋白的表达。 3. PFC可能通过增加肺泡液体清除相关离子通道ENaC-α、γ,Na~+-K~+-ATPase-α1,β1和CNG1-α mRNA和ENaC-α和Na~+-K~+-ATPase-α1蛋白表达及增强Na~+-K~+-ATPase酶活性，以降低LPS对肺泡上皮液体清除能力的损伤。
[Abstract]:Objective in ALI / ARDS patients, the expression and functional activity of alveolar fluid clearance related ion channels decreased, the alveolar fluid clearance ability was impaired, and the accumulation of fluid in alveolar cavity was increased, which was the direct cause of respiratory distress and was related to the poor prognosis of the disease. By improving alveolar fluid clearance, it may be a new way to cure ALI / ARDS in the future. The aim of this study was to investigate the protective effect of perfluorocarbon on lipopolysaccharide-induced alveolar fluid clearance related ion channel expression and function down-regulation. Methods (1) PFC-in-DMEN was prepared by ultrasonic cell fragmentation instrument, PFC-in-DMEMN with different volume ratio (PFC-in-DMEMN) was used to treat A549 cells for 0.5 h, 1 h and 2 h for 4 h, and the optimal concentration and time of PFC treatment were optimized (P < 0.05). LPS10ugrml) was used to interfere with A549 cells to establish lung injury cell model. The cells were treated with PFC-in-DMEM10ugml / ml and PFC-in-DMEM ~ LPS-10ugP / ml for 4 h respectively. The protein expression of ENAC- 伪, 尾, 纬 Na-K-ATPase- 伪, 尾 _ 1CNG1- 伪 and AQP3 mRNA were detected by real-time quantitative PCRQPCR- 伪, 尾 _ 1CNG1- 伪 and AQP3 mRNA, and the protein expression of ENaC- 伪 Na ~ -K ~ -ATPase- 伪 _ 1 was analyzed by cell Na ~ -K ~ -ATPase activity assay kit. Results the highest level of ENAC- 伪 and Na ~ -K~ -ATPase- 伪 _ 1 mRNA expression was observed in A549 cells treated with PFC for 4 h. The increase of 1L-1 尾 -IL-6 and TNF- 伪 mRNA expression in A549 cells reached its peak at 6 h and decreased at 4 h. However, the mRNA of ENAC- 伪 and Na ~ -K- ATPase- 伪 _ 1 increased slightly at 0.5 h after treatment with LPS, but there was no significant difference in the decrease of ENAC- 伪 and Na ~ -K- ATPase- 伪 _ 1 mRNA for 4 h (P 0.037%, P 0.046%, respectively). (3) compared with the control group, the mRNA expression of ENaC- 伪, 尾 -Na ~ -K ~ -ATPase- 伪 _ 1, 尾 _ 1 in LPS group and the expression of ENAC- 伪 -K ~ -ATPase- 伪 _ 1 protein in LPS group were significantly decreased, but the mRNA expression of ENAC- 纬 -CNG1- 伪 and AQP3 were not significantly decreased in LPS group. Compared with LPS, PFC ~ LPS could significantly up-regulate the mRNA expression of ENAC- 伪, 纬 -Na ~ -K ~ -ATPase- 伪 _ 1, 尾 _ 1 and CNG1- 伪. The protein expression of ENAC- 伪 -Na ~ -K ~ -ATPase- 伪 _ 1 was upregulated significantly. Compared with LPS, PFC ~ LPS could significantly upregulate the activity of Na ~ -K ~ -ATPase for 4 h. Conclusion 1.10ug/ml can induce the production of proinflammatory cytokines such as IL-1 尾 -IL-6 and TNF- 伪 in A549 cells, and make alveolar fluid clear ENaC- 伪, 尾 -Na ~ -K ~ -ATPase- 伪 1, 尾 1 mRNA expression and decrease the expression of ENAC- 伪 Na ~ -K ~ -Pase- 伪 1 protein in A549 cells at 6 h, indicating that the ALI cell model is successful. And can be used in Ali alveolar fluid clearance of related cells. 2. PFC could directly increase the expression of ENaC- 纬 and CNG1 伪 mRNA and protein of ENAC- 伪 and Na-K- ATPase- 伪 in alveolar fluid scavenging channels of normal A549 cells. 3. PFC may increase the expression of ENaC- 伪, 纬 -Na ~ -K ~ -ATPase- 伪 _ 1, 尾 _ 1 and CNG1- 伪 mRNA and the protein expression of ENAC- 伪 and Na ~ -K ~ -ATPase- 伪 _ 1, and enhance the activity of Na ~ -K ~ -ATPase enzyme in alveolar epithelial fluid clearance by increasing alveolar fluid scavenging channels, so as to reduce the injury of LPS to alveolar epithelial fluid clearance.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R563.8

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